Project Summary Gram negative bacteria secrete outer membrane vesicles (OMVs), which deliver a wide array of biomacromolecules, including toxins and virulence factors to host cells. Aggregatibacter actinomycetemcomitans (Aa) OMVs are enriched in leukotoxin (LtxA) and have been observed to contain lipids that are not found in the bacterial outer membrane (OM). The mechanism leading to LtxA enrichment in OMVs has not yet been identified. We have observed that LtxA has a strong affinity for lipopolysaccharide (LPS), the primary component of the outer leaflet of the OM. In several organisms, variations in the LPS structure within the OMVs has been found to differ from that of the OM. We therefore hypothesize that, in Aa, the OMV-specific lipids are LPS variants and that the differential affinity of LtxA for these variants regulates the toxin's enrichment in the OMV. To study this process, we will determine the LPS and LtxA composition of both the OMV and OM of Aa. We will conduct this series of experiments using OMVs purified from Aa that has been grown under a variety of conditions to obtain a better picture of LtxA enrichment in LPS under biologically relevant conditions. We will then purify the LPS from Aa OMVs and OM and quantify the affinity of LtxA for each type of LPS to relate affinity of the toxin for LPS to its observed enrichment. We anticipate that the research will result in a more complete understanding of the role of OMVs in periodontitis. In addition, the results of the work may be more broadly applicable to the general understanding of protein enrichment in or exclusion from OMVs.